Abstract

Interaction forces between biological molecules such as antigen and antibody play
important roles in many biological processes, but probing these forces remains technically
challenging. Here, we investigated the specific interaction and unbinding forces between
human IgG and rat anti-human IgG using self assembled monolayer (SAM) method for sample
preparation and atomic force microscopy (AFM) for interaction force measurement. The
specific interaction force between human IgG and rat anti-human IgG was found to be
0.6–1.0 nN, and the force required for unbinding a single pair of human IgG and rat
anti-human IgG was calculated to be 144 ± 11 pN. The results are consistent with those
reported in the literatures. Therefore, SAM for sample preparation combined with AFM
for interaction measurement is a relatively simple, sensitive and reliable technique
to probe specific interactions between biological molecules such as antigen and antibody.

Keywords:

Introduction

Structure, dynamics and function of biological molecules are largely determined by
physical forces acting on and between the molecules. For example, the intermolecular
adhesion force between an antigen and an antibody essentially determines whether the
two molecules recognize and bind to each other to initiate a response in the immune
system. Therefore, measurement of interaction forces between biological molecules
is important to elucidating single molecule recognition processes such as antigen–antibody
binding and unbinding, ligand-receptor attachment and activation [1-3]. Several techniques have been developed for measuring interaction forces between
biological molecules including surface forces apparatus (SFA), optical or magnetic
tweezers and atomic force microscopy (AFM) [4-7]. SFA is a classical technique with high sensitivity to small interaction forces,
but also with significant limitations such as being technically demanding, only applicable
to surfaces of large area [4,5]. More recent techniques are optical or magnetic tweezers and AFM [7]. The latter has emerged as widely used because (1) most reported single-molecule
interaction forces have fallen well within the measuring range of AFM [2,8,9], (2) AFM can measure interaction forces under near physiological conditions with
high resolution of both force and space [10,11], (3) with functionalized measuring tip, AFM is capable of sensing and mapping interaction
forces across a large area such as the entire surface of a living cell [6,12-15].

One of the major challenges, however, for using AFM to measure interaction force between
biological molecules is the sample preparation, in particular, the coating of molecules
onto the surface of the substrate, and the AFM tip (functionalizing AFM tip). Currently,
an extensively used method is to chemically link biological molecules onto the surface
either by silanization or by thiol-based self-assembled monolayers (SAM) [16,17]. SAM has been developed over two decades ago and proven to be an effective and facile
way to form well-defined and controlled films. Here, we demonstrate that by employing
SAM method, rat anti-human immunoglobulin G (IgG) and human IgG could be linked onto
the surface of gold substrate, and AFM tip, respectively, and used as a model system
to probe antibody–antigen interaction forces by AFM. The single-molecule-specific
adhesion force between human IgG and rat anti-human IgG was further calculated by
Poisson statistical method. The results suggest that this method may provide a relative
simple and reliable way to probe specific interactions between biological molecules.

Methods and Materials

The simple mechanism for immobilizing proteins such as antibodies onto thiol-based
SAM has been described by Ferretti et al. [17]. Briefly, sulfur-containing molecules (thiols, sulfides and disulfides) have a strong
affinity for gold and will interact with it in near covalent manner. Therefore, when
gold is immersed into a solution of thiols such as 16-Mercaptohexadecanoic acid (MHA),
the thiol molecules will spontaneously react with gold and form a SAM of thiols on
the gold surface with tightly packed and well-ordered chains. The terminal end of
the thiol-based SAM consists of carboxyl tail groups that can be activated by the
1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide
(NHS). The activated SAM can then be soaked into protein solution to form protein
layer.

Gold-Coated Substrate

Gold-coated substrate was prepared by vapor deposition of gold onto mica substrate
that had been freshly cleaved and preheated to 325 °C for 2 h by a radiator heater.
The high vacuum evaporator in which the gold was vapor deposited onto mica substrate
was operated at the pressure of ~10−7 Torr, and evaporation rate of 0.1–0.3 nm/s, resulting in a final thickness of the
coated gold film at ~200 nm. A chromium layer was also vapor deposited and sandwiched
between the gold and mica to strengthen the adhesion between the surfaces. The gold-coated
substrate was then annealed in H2 flame for 1 min before use.

SAM of Thiols on Gold Surface

The bare gold-coated substrate prepared as above was thoroughly cleaned in hot piranha
solution (v/v H2SO4:H2O2 = 3:1) for 30 min. The gold-coated substrate was then immersed into the ethanol solution
of 1 mM MHA for 24 h to produce the thiol-based SAM on the gold surface (Fig. 1 left panel, columns 1–2), and unbound thiols were removed by ultrasonication in pure
ethanol for 2 min. The prepared SAM was then rinsed sequentially with pure ethanol,
ultra pure water, and finally dried in a N2 stream before use.

Figure 1. A schematic illustration of the methodology used in this study. From left to right, the gold-coated surface of substrate was first modified by soaking it into the ethanol
solution of 16-Mercaptohexadecanoic acid (MHA) for 24 h, which formed an MHA film
on the surface of gold substrate. Then, the MHA-modified surface of gold substrate
was subject to NHS and EDC in PBS solution for 1 h to activate the MHA film. Afterward,
the activated MHA film on the gold substrate was immersed into protein (rat anti-human
IgG in this case) solution at 4 °C for 8 h, resulting formation of a well-defined
protein monolayer on the MHA-modified gold substrate. Similarly, protein (human IgG
in this case) monolayer was formed on an AFM tip. The antigen-functionalized AFM tip
scanned across the well-ordered antibody monolayer, and at a number of randomly chosen
locations the force–displacement curves between the tip, and the substrate surface
were recorded and transformed into force–displacement curves by AFM

Protein Immobilization onto the SAM

Protein immobilization was carried out according to the method published by Wakayama
et al. [18] with minor modification. In brief, the thiol-based SAM was treated in the solution
of 2 mg/mL NHS and 2 mg/mL EDC in PBS for 1 h, which activated the carboxylic acid
terminal groups of the SAM (Fig. 1 left panel, column 3). After thoroughly rinsed with ultra pure water, and dried in
N2 stream, the activated SAM was then immersed into the protein solution of 7 μg/mL
rat anti-human IgG in PBS and incubated at 4 °C for 8 h to immobilize the proteins
onto the SAM (Fig. 1 left panel, column 4). The prepared sample of protein layer was stored in PBS at
4 °C before use.

Functionalization of AFM Tip

Functionalized AFM tip with human IgG coating was prepared similarly as described
above. First, the AFM tip was cleaned in the hot piranha solution for 30 min and then
rinsed with ultra pure water. Subsequently, the tip surface was coated with thiol-based
SAM in the solution of MHA and then activated in the solution of EDC and NHS. Finally,
the tip was functionalized with human IgG coating by incubating the activated tip
in PBS solution of the protein at concentration of 7 μg/mL, at 4 °C for 8 h. The functionalized
tip was stored in PBS at 4 °C before use.

Measurement of Antigen–Antibody Adhesion Force by AFM

Adhesion force between human IgG and rat anti-human IgG was measured by AFM using
Benyuan CSPM 5000 scanning probe microscope (Benyuan Co., China). As shown in the
right panel of Fig. 1, the functionalized AFM tip scanned across the well-ordered protein monolayer. At
a given location, the tip was moved toward the surface of the monolayer and retracted.
When the tip approached the monolayer surface, it would deflect due to the antigen–antibody
interaction force, which would be detected as a “voltage-displacement” signal and
converted into a “force–displacement” curve [4,13,19]. Because the tip was considered an elastic cantilever, its deflection was determined
by the force (F) exerted on it following Hooke’s law, i.e., F = k × d, where d is the deflection, k is the spring constant of the cantilever tip. Generally, k should be small for AFM in order to minimize measurement noise [4]. In this study, commercially available gold-coated Si3N4 cantilever tip (BudgetSensors ®, Innovative Solutions Bulgaria Ltd. Bulgaria) was used of which the spring constant,
calibrated by thermal fluctuation method [20], was 0.2–0.3 N/m. The tip has a pyramidal geometry, its tip radius is about 25 nm,
and the thickness of the gold layer is 70 nm.

All force measurements were taken by using contact mode AFM with PBS as the medium
between the tip and the protein monolayer, and the retraction velocity was estimated
to be 0.04 μm/s. From the “force–displacement” curve, the adhesion force between the
rat anti-human IgG on the substrate and the human IgG on the tip was calculated. Measurement
was repeated many times at each of several randomly selected locations across the
protein monolayer on the gold substrate.

Specificity of the Measured Adhesion Force

In order to consider specific adhesion force only, any nonspecific interaction force
between the human IgG and the rat anti-human IgG should be measured and excluded.
This was done by a blocking experiment performed as follows. First, the AFM tip coated
with human IgG was incubated for 30 min in solution of rat anti-human IgG to block
the binding sites of the antigen on the tip. Then, the nonspecific interaction force
was obtained by the same force measurement as described above, but performed using
the blocked tip.

Results and Discussion

Although SAM method is relatively simple and easy to do, there are many aspects that
need to be considered carefully in order to form a satisfactory protein monolayer
on SAM-modified substrate [16,17,21,22]. These include, but not limited to, the following: (1) gold was used as substrate
because it is chemically inert, and thiols bind to it with a high affinity; (2) MHA
was used to form thiol-based SAM because of its flexible long carbon chain that served
as a spacer to minimize interference between the protein molecules and the gold substrate;
(3) protein immobilization was carried out in PBS at 4 °C and pH = 7.4 because that
pH and temperature may both affect protein activity; (4) the coated protein layer
should not only provide optimally orientated protein molecules, but also give minimal
steric hindrance to the protein molecules so that they can mimic their natural state;
(5) in addition to that 1 mM thiol concentration and 24 h immersion that were sufficient
for forming well-ordered SAM of thiols [16], the protein concentration was also important for forming uniform protein monolayer.
We found that 10 μg/mL was the adequate protein concentration for forming uniform
layer, and above this concentration the proteins might aggregate and form irregular
layer. Considering that SAM method has been proven capable of ensuring the activity,
mobility and stability of protein molecules [10,16], and all experimental aspects addressed properly as described above, the method presented
here can be used to prepare reliable sample surface of biological molecules for AFM
force measurement. Indeed, the topography of protein-modified surface prepared using
this method had been examined by AFM imaging and confirmed satisfactory [23].

Figure 2 shows three representative force–displacement curves obtained by AFM measurement
between rat anti-human IgG monolayer formed on thiol-based SAM substrate and (1) original
bare tip, (2) blocked tip prepared as described in “Specificity of the Measured Adhesion
Force”, (3) tip coated with human IgG. These force–displacement curves characterize
the binding and unbinding events between the AFM tip and the substrate when there
were either no interactions, only nonspecific interactions, or specific interactions,
respectively. The binding force and its probability distribution were calculated from
repeated measurements and plotted in Fig. 3. The results demonstrate that, considering the noise floor of the measurement, there
were no interaction forces between the bare tip and rat anti-human IgG on the substrate.
When the antigen-coated tip was blocked, there were no interactions for most of the
time, but occasionally (approximately 20% probability) there were small interaction
forces occurring between the tip and rat anti-human IgG on the substrate. These occasional
small binding could be attributed to `olecules [24,25]. In contrast to these conditions, when the tip was coated with human IgG, there were
marked binding forces measured between the human IgG and rat anti-human IgG. Although
the magnitudes (refer to the maximal downward cantilever deflection during a retraction
curve compared to the baseline) of measured interaction forces spread from 0.2 to
1.8 nN, the majority of them were between 0.6 and 1.0 nN. The variation of the measured
interaction forces between the antigen and antibody could be attributed to the variation
of contact areas between the tip and the protein monolayer when probed at different
time and different locations, the density distribution of protein molecules on the
substrate, and thermal fluctuation of AFM [26,27]. The loading rate of force measurement might also contribute to the variation of
measured binding force values [18,28]. In this study, the retraction velocity was estimated to be 0.04 μm/s, all measurements
were observed under this condition.

Figure 2. Three typical force–displacement curves of AFM are shown to demonstrate the interaction
events between the tip and the substrate surface. a–c The force–displacement curves when there were either no interactions (bare tip/rat
anti-human IgG), specific interactions (human IgG/rat anti-human IgG), or only non-specific
interactions (blocking experiment)

Since the contact area of AFM tip is very large relative to the size of protein molecule
attached to it, there had to be multiple pairs of antigen–antibody involved during
each single interaction event detected by AFM. Thus, the interaction force measured
by AFM was not that of a single antigen–antibody pair, but rather a collective result
of interaction forces from multiple antigen/antibody pairs. However, the Poisson statistical
method developed by Beebe et al. could be used to determine the unbinding force required
to separate a single pair of antigen and antibody molecules [29-31]. The principal assumption of this method is that during each unbinding event as an
AFM tip is pulled off the substrate, the number of antigen–antibody pairs that contribute
to the total adhesive force is finite and, more importantly, follows a Poisson distribution
when the unbinding event is observed repeatedly within the same fixed area of contact.
The advantage of this method is that it provides an accurate calculation of single-molecule
adhesive force in the presence of moderate-to-large variation or noise of various
types [32]. As defined by the Poisson distribution, the mean value equals the variance of the
number (n) of interacting antigen–antibody pairs. Provided that the measured total adhesion
force is composed of a finite number of discrete interacting antigen–antibody pairs
within a fixed contact area, the adhesion force between a single antigen–antibody
pair (Fi) and possible nonspecific interaction force (F0) can be derived from the slope and interception of the linear regression curve of
the variance (σm2) versus the mean (μm) of the measured total adhesion force as [29].

The total adhesion force between human IgG and rat anti-human IgG were measured repeated
for 50–55 times at each of several randomly chosen locations of the rat anti-human
IgG monolayer, and the mean (μm) and variance (σm2) of these measurements are given in Table 1, and plotted with linear regression as shown in Fig. 4. From these results, the specific adhesion force between a single pair of human IgG
and rat anti-human IgG,Fi and the nonspecific force,F0, were calculated as 144 ± 11 and 69 pN, respectively. This level of specific adhesion
force was well within the range of 35–165 pN that has been reported as the estimated
range of force required to rupture a single antigen–antibody complex [33]. Comparison among several antibody-based single molecular interaction forces is summarized
in Table 2. The specific interaction force value of human IgG/rat anti-human IgG was close to
that of collagen/collagen antibody, but significantly lower than that of anti-angiogenin
antibody IgG/angiogenin. The specific forces are largely influenced by the tip-sample
system, the surface properties and the experimental setup. For example, the same molecular
partners, namely human serum albumin (HSA) and anti-HSA, were measured by Hinterdorfer
et al. [34] and Idiris et al. [38], they used different methods to immobilize the protein molecules, placed the antibody
molecules on the contrary position (tip or substrate), the resulting force values
were significantly different. Since in the Poisson distribution method, the chemical
bonds, hydrogen bonds and van der Waals force are considered as specific interactions
yielding a total adhesion force, in this case, it is difficult to define or attribute
the measured nonspecific force to certain forces.

Table 1. Unbinding forces between human IgG and rat anti-human IgG measured at five different
locations

Figure 4. The variance (σm2) was plotted versus the mean (μm) of the measured interaction forces between human IgG and rat anti-human IgG. Each
data point represents a data set taken at one of the five different sample locations
(R = 0.9907). Details of the data sets are given in Table 1

Table 2. Comparison of several measurements of antibody-based protein–protein interactions
by AFM

Conclusions

Protein monolayers of rat anti-human IgG and human IgG were covalently bound on gold
substrate and AFM tip using SAM method. Thus, the interactions between the antigen
and antibody molecules on the gold substrate and the AFM tip were probed by atomic
force microscopy. The specific interaction forces were determined to be largely within
a range of 0.6–1.0 nN. Moreover, based on these measurements and the Poisson statistical
method, it was calculated that the force required for unbinding a single antigen/antibody
pair was 144 ± 11 pN, and the nonspecific interaction force was 69 pN, respectively.
These results are consistent with those measured by other, more complex methods, and
suggest that when SAM method is properly used to prepare the sample surfaces, AFM
can be a relatively simple, sensitive and reliable technique to probe specific interactions
between biological molecules such as antigen/antibody pairs.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 30670496,
30770529) and the Scientific Research Foundation for the Returned Overseas Chinese
Scholars, State Education Ministry (2006-331) and the Natural Science Foundation Project
of CQ CSTC (2006BB5017).

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